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TRANSCRIPT
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ANTIBACTERIAL ACTIVITY IN MEDICINAL PLANTS
Dissertation
Submitted to the University of Kerala
For the partial fulfillment of the requirements for the award of
Bachelor’s degree in Biotechnology
By
Iype Praveen Thomas , Unnimaya .S , Vandana Balakrishna , NeethuNath.R.S , Sreelekshmi.S , Laya Johnson ,Sherin .P.L, Sumita Muralidhar.
Department of Botany and Biotechnology
Mar Ivanios College
Bethany Hills
Trivandrum – 15
2011-2012
Thiruvananthapuram-15
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CERTIFICATE
This is to certify that this dissertation entitled
“ANTIBACTERIAL ACTIVITY IN MEDICINAL PLANTS” is an authentic record of
the studies and research work carried out by Mr. /Ms ……………… under my supervision
and guidance for the partial fulfillment of the requirements for the degree of Bachelor of
Science in Biotechnology (Restructured) of the University of Kerala and that no part of this
dissertation has been presented earlier for any degree or diploma in this or any Universities.
MrsThressiamma.Mathew Dr.Bindhu.T.Alex
Head of the department Supervising teacher
Examiners:
1.
2.
3
Declaration
We hereby declared that this dissertation entitled “ANTIBACTERIAL ACTIVITY IN
MEDICINAL PLANTS” is an authentic record of the orginal research work carried out by
us under the supervision of Dr. Bindhu . T. Alex, Department of Botany & Biotechnology,
Mar Ivanios College, Thiruvananthapuram and that not part of this dissertation has been
previously presented for any degree or diploma or similar title of this or any other
Universities.
Iype Praveen Thomas (62436)
Unnimaya.S (62434)
Vandana Balakrishna (62413 )
Neethu Nath .R.S (624 )
Sreelekshmi.S (62435)
Laya Johnson (624 )
Sherin .P.L (62425)
Sumita Muralidhar (62412 )
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ACKNOWLEDGEMENT
We are very much obliged to Dr. Bindhu .T.Alex , Assistant Professor, Department of
Botany & Biotechnology, Mar Ivanios College, Nalanchira for suggesting this topic and also
for guiding us throughout the course of this dissertation. We also have the good fortune to be
able to draw upon the devotion, deep knowledge and mature experience of his for the
completion of this study.
We wish to express our heartfelt gratitude and indebtedness to Mrs. Thressiamma Mathew,
Head of the Department, Department of Botany & Biotechnology, Mar Ivanios College,
Nalanchira for providing the infrastructure facilities for this dissertation. We take this
occasion to thank Ms. Fathima Zehra (Department of Botany & Biotechnology) for being
with us during this work.
We express our heartfelt thanks to Mr. R.Surendran, Laboratory Assistant (Department of
Botany & Biotechnology for the help he had rendered in the laboratory. We are grateful to all
our classmates for their co-operation and suggestion. Moreover we are greatly indebted to our
parents for their encouragement and support.
Above all we are deeply indebted to “The God Almighty” who always remains as the
helping hand behind us in all crises.
Iype Praveen Thomas (62436)
Unnimaya.S (62434)
Vandana Balakrishna (62413 )
Neethu Nath .R.S (624 )
Sreelekshmi.L (62433)
Laya Johnson (624 )
Sherin .P.L (62425)
Sumita Muralidhar (62412 )
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CONTENTS
INTRODUCTION
OBJECTIVE
REVIEW OF LITERATURE
MATERIALS AND METHODS
RESULTS AND DISCUSSION
SUMMARY AND CONCLUSION
BIBLIOGRAPHY
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INTRODUCTION
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Medicinal plants are an integral component of ethenoveterinary
medicine. Medicinal plants, since time immemorial, have been used virtually all cultures as a
source of medicine. The widespread use of herbal remedies and healthcare preparations, as
those described in ancient texts, and obtained from commonly used traditional herbs and
medicinal plants, has been traced to the occurrence of natural products with medicinal
properties. The use of traditional medicine and medicinal plants in most developing countries,
as a normative basis for the maintenance of good health, has been widely observed.
As defined by WHO, health is a state of complete physical, mental
and social wellbeing and not merely the absence of disease or infirmity.Medicine, in several
developing countries, using local traditions and beliefs, is still the mainstay of healthcare. The
practice of traditional medicine is widespread in China, India, Japan, Pakistan, Srilanka and
Thailand. In China, about 40% of the total medicine consumption is attributed to traditional
tribal medicines. In Thailand, herbal medicines make use of legumes encountered in the
Caesalpiniaceae, the Fabaceae and the Mimosaceae.
Ethno botanical information is leading to the discovery of novel
phytopharmaceuticals and other phytoproducts.This trend has made their commercialization a
necessity.Therfore industries based on medicinal and aromatic plants have been established
allover the world with a view to manufacture the so-called green products to satisfy the
growing demand. The development of formulation of drugs of plant origin involves botanical
identification of the vegetable drug,cultivation and post harvest procedures, extraction
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procedures, standardization of extracts and pharmaceutical formulations.This means that the
phytotherapeutics are in the hands of personnel from different disciplines. The production of
phytotherapeutics and drugs from the plants needs the cooperation of a big team of
horticulturists, botanists,
ecologists,taxonomists,phytochemists,pharmacists,pharmocologists,pharmaceutical
specialists, marketing and distribution specialists.
HISTORY OF MEDICINAL PLANTS
As early as 3000BC,the ancient Egyptians put much confidence in
plants for curing many diseases.Until now the same confidence is still existing among the
contemporary Egyptians and a “turn back”to “remedy by herbs”is now becoming global
rather than regional or national request.This is strongly favoured,since the natural drugs have
little more or no side effects as do the chemically synthesized medications.
Pharmacopoeial wild medicinal plants: These plants are used in folk medicine
since a long time ago.Recent and modern studies on these plants proved the occurance of
active principles in them. Their pharmacological activity has been investigated.They are
among the pharmacopoeial drugs in in different pharmaopoeias;either in thevArab countries
or abroad.
Plants used in folkmedicine:There are numerous plant species which are
collected from the field to be sold in the “Attarin:”or the herb seller shops.
Plants of potential medicinal value:Many plant species were investigated for
their active constituents.These has been done depending on the information of the folk use of
these plants,or in species with relatives of species,genera or the same family,known from
other countries to have active constituents.
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Medicinal plants are an important health and economic component of the
floras in the developed as well as developing countries.Increasing worldwide interest in
herbal remedies expanding reliance of local health care of traditional remedies,and a renewed
interest in the development of pharmaceuticals from plant sources have greatly increased
trade in medicinal plant materials.Important populations of medicinal plants are found not
only in the regions and ecosystems with high biological diversity but also in less diverse
floras and in floristic communities that are not a common focus of conservation efforts.
The production of medicinal and aromatic plants requires an
inderstanding of plant growth,ecology,business,economics,law,conservation and a lot of
subjects related to tillage and gathering plants.While development such as
machinery,fertilizers and pesticides have helped farmers meet demands for quality materials
at affordable prices,the balance with farming costs,and labour compel societies to set
directions and establish limits.
Plants produce a diverse range of bioactive molecules,making them a
rich source of different types of medicines.Higher plants,as sources of medicinal compounds,
have continued to play a dominant role in the maintainance of human health in ancient times.
Over 50% of all modern clinical drugs are of natural product origin and natural products play
an important role in drug development programs in the pharmaceutical industry. The
relatively lower incidence of adverse reactions to plant preparations compared to modern
conventional pharmaceuticals,coupled with their reduced cost, is encouraging,both the
consuming public and national health care institutions to consider the plant medicines as
alternatives to synthetic drugs. Plants with possible antimicrobial activity should be tested
against an appropriate microbial model to confirm the activity to ascertain the parameters
associated withit.The effects of plant extracts on bacteria have been studied by a very large
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number of researchers in different parts of the world. Much work has been done on ethno
medicinal plants in India.
Infectious diseases account for approximately one half of all deaths
in tropical countries. In industrialized nations, despite the progress made in the understanding
of microbiology and their control,incidents of epidemics due to drug resistant micro
organisms and the emergence of hither to unknown disease causing microbes,pose enormous
public health concerns.Historically plants have provided a good source of antiinfective
agents;emetine,quinine,and berberine remain highly effective instruments in the fight against
microbial infections.Phytomedicines derived from plants have shown great promise in the
treatment of intractable infectious diseases including opportunistic AIDS.
Historically,plants have provided a source of inspiration for novel
drug compounds,as plant derived medicines have made large contributions to human health
and well being.Their role is twofold in the development of new drugs: (1)they may become
the base for the development of a medicine,a natural blueprint for the development of new
drugs or (2) a phytomedicine to be used for the treatment of disease.
The first generation of plant drugs were usually simple botanicals
employed in more or less their crude form.Several effective medicines used in their natural
stale such as chinchona,opium,belladonna and were selected as therapeutics agents based on
empirical evidence of their clinical application by traditional societies from different parts of
the world.Folllowing the industrial revolution,a second generation of plant based drugs
emerged based on scientific processing of the plant extracts to isolate “their active
constituents.”
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The second generation phytopharmaceutical agents were pure
molecules and some of the compounds were even more pharmacologically active than their
synthetic counterparts. Notable examples were Quinine from Chinchona,reserpine from
Rauvolfia and more recently taxol from Taxus species.These compounds differed from the
synthetic therapeutic agents only in their origin. They follow the same method of
development and evaluation as other pharmaceutical agents.In the development of “Third
Generation”phytotherapeutic agents a top-bottom approach is usually adopted.This consists
of conducting a clinical evaluation of the treatment modalities and therapy as administrated
by traditional doctors or as used by the community as folk medicine.This evaluation is
followed by acute and chronic toxicity studies in animals.Studues should,when
applicable,includes cytotoxicity studies. It is only if the substance has an acceptable safety
index would it be necessary to conduct detailed pharmacological/biochemical studies.
PRESENT USE OF PLANTS
It is estimated that today,plant materials are present,in or have provided
the models for 50%Western drugs.Many commercially proven drugs used in modern
medicine were initially used in crude form in traditional or folk healing practices,or for other
purposes that suggested potentially useful biological activity.The primary benefits of using
plant derived medicines are that they are relatively safer than synthetic alternatives,offering
profound therapeutic benefits and more affordable treatment.
NEEM
For thousands of years the beneficial properties of Neem (Azadirachta indica A. Juss) have
been recognized in the Indian tradition. Each part of the neem tree has some medicinal
property. Biswas et al (2002) have recently reviewed the biological activities some of the
neem compounds, pharmacological actions of the neem extracts, clinical study and plausible
medicinal applications of neem along with their safety evaluation.
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Neem has two closely related species: A. indica A. Juss and M. azedarac, the former
is popularly known as Indian neem (margosa tree) or Indian lilac, and the other as the Persian
lilac. Neem has been extensively used in ayurveda, unani and homoeopathic medicine.The
Sanskrit name of neem tree is Arishtha meaning 'reliever of sickness' and hence is considered
asSarbaroganibarini. The tree is still regarded as 'village dispensary' in India. The
importance of the neem tree has been recognized by US National Academy of Sciences,
which published a report in 1992 entitled 'Neem – a tree for solving global problems'.
More than 135 compounds have been isolated from different parts of neem and several
reviews have also been published on the chemistry and structural diversity of these
compounds. The compounds have been divided into two major classes: isoprenoids (like
diterpenoids and triterpenoids containing protomeliacins, limonoids, azadirone and its
derivatives, gedunin and its derivatives, vilasinin type of compounds and C- secomeliacins
such as nimbin, salanin and azadirachtin ) and non-isoprenoids, which are proteins (amino
acids) and carbohydrates (polysaccharides), sulphurous compounds, polyphenolics such as
flavonoids and their glycosides, dihydrochalcone, coumarin and tannins, aliphatic
compounds, etc.
Various parts of the neem tree have been used as traditional Ayurvedic medicine in India.
Neem oil and the bark and leaf extracts have been therapeutically used as folk medicine to
control leprosy, intestinal helminthiasis, respiratory disorders, constipation and also as a
general health promoter. Its use for the treatment of rheumatism, chronic syphilitic sores and
indolent ulcer has also been evident. Neem oil finds use to control various skin infections.
Bark, leaf, root, flower and fruit together cure blood morbidity, biliary afflictions, itching,
skin ulcers, burning sensations and pthysis.
Oil from the leaves, seed and bark possesses a wide spectrum of antibacterial action against
Gram-negative and Gram-positive microorganisms, including M. tuberculosis and
streptomycin resistant strains. In vitro, it inhibits Vibrio cholerae ,Klebsiella pneumoniae, M.
tuberculosis and M. pyogenes. Antimicrobial effects of neem extract have been demonstrated
against Streptococcus mutans and S. faecalis.
Neem extract
Its effective to cure ringworm, eczema and scabies. Lotion derived from neem leaf, when
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locally applied, can cure these dermatological diseases within 3-4 days in acute stage or a
fortnight in chronic case. A paste prepared with neem and turmeric was found to be effective
in the treatment of scabies in nearly 814 people100.
Neem leaf extract has been prescribed for oral use for the treatment of malaria by Indian
ayurvedic practitioners from time immemorial. Recently, a clinical trial has been carried out
to see the efficacy of neem extract to control hyperlipidemia in a group of malarial patients
severely infected with P. falciparum. The lipid level, especially cholesterol, was found to be
lower during therapy when compared to non-malaria patients. Reports are available regarding
the use of neem to treat patients suffering from various forms of cancer. One patient with
parotid tumour and another with epidermoid carcinoma have responded successfully when
treated with neem seed oi1.
NIM- 76, a refined product from neem oil, was studied in 10 human volunteers, where intra-
vaginal application before sexual intercourse could prevent pregnancy with no adverse effect
on vagina, cervix and uterus. The data suggested that intrauterine treatment is safe.
KACHOLAM
Kacholam ( Kaempferia galanga L). is a highly useful medicinal plant. Its rhizomes (tubers)
are used in indigenous medicines as stimulant, expectorant, diuretic, carminative etc.
Decoction of rhizomes is useful for curing dyspepsia, headache, rheumatism and malaria. For
clearing nasal obstructions, rhizomes may be boiled in oil may be applied externally.
Digestive power can be enhanced by using kacholam tubers. Larvicidal and anticancerous
principles are extracted from tubers. The tubers enhance digestion, remove bad odour of
mouth and are used as masticatory for chewing along with betel and arecanut .
Kacholam, also known as Sugandhavacha, Chandramulika or Sidhul is a rhizomatous
perennial plant, the rhizomes of which yield an essential oil. The oil is utilized in the
manufacture of perfumes and in curry flavouring. It is also employed in cosmetics, mouth-
washes, hair tonics and toiletries. The pungent, hot, sharp, bitter and aromatic rhizomes find
an important place in indigenous medicine as stimulant, expectorant, diuretic and
carminative. It promotes digestion and cures skin diseases, piles, phantom tumors, coughs,
oedema, fever, epilepsy, splenic disorders, wounds, asthma and rheumatism. The rhizomes
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are used for protecting clothes against insects and are eaten along with betel and arecanut as a
masticatory. The rhizomes and leaves are attached to neck- laces and added to bath water for
perfume.Karcura (Kacholam) is a reputed remedy for all diseases caused by the morbidity of
vata and kaphaand is especially useful in respiratory ailments like cough, bronchitis and
asthma. The drug is reported to be acrid, hot, bitter and aromatic. It cures skin diseases,
wounds and splenic disorders. It promotes digestion, removes bad odour of the mouth and
destroys pathogenic organisms (Aiyer and Kolammal, 1964). The officinal part is the rhizome
and it is a constituent of a variety of Ayurvedic preparations like Dasamularista, Valiya
rasnadi kasaya, kaccoradi churna, Asanaeladi taila. Valiya narayana taila, etc. The identity of
the plant sources of the drug Karcura is a matter of debate. The source of Karcura in Kerala
in the recent times has been Kaempferia galanga (Sivarajan and Balachandran,
1994).
The rhizomes and root-stocks are good for dyspepsia, leprosy, skin diseases, rheumatism,
asthma, cough, bronchitis, ulcers, helminthiasis, fever, malarial fever, splenopathy,
cephalalgia, inflammatorytumour, nasal obstruction, halitosis, strangury, urolithiasis, and
haemorrhoids. The leaves are used for pharyngodynia, ophthalmia, swellings, fever and
rheumatism . The tubers reduced to powder and mixed with honey are given in case of
coughs and pectoral infections. The oil in which they are boiled is useful in applying to the
stoppages of the nasal organs . Kaempferia galanga is reported to have great export
potential . The tuberous rhizome of kacholam contains an alkaloid, starch, gum, fatty matter
with a fragrant liquid essential oil and a solid white crystalline substance and mineral matter.
The rhizome possesses a camphoraceous odour with somewhat bitter aromatic taste
resembling that of Hedychium spicatum.The essential oil is reported to contain over 54
components of which the major constituents are ethyl-trans-p-methoxy, cinnamate 16.5%,
pentadecane 9%, 1,8-cineole 5.7%, g-carene 3.3%, and borneol .Insectididal constituents
from Kaempferia galanga were isolated by .Cyclohexane oxide derivatives and diterpenes
were isolated by . Rhizome yields essential oil, which has antifungal activity. Ethyl-p-Methyl
O-trans-cinnamate is the main compound in root . p-Methoxycinnamic acid and its methyl
and ethyl esters have been isolated from the essential oil . Essential oil from rhizomes contain
npentadecane, ethyl-p-methoxy cinnamate, ethyl cinnamate, carene, camphene, borneol, p-
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methoxystyrene, pmethoxy cinnamate, p-methoxy- trans-cinnamic acid and cinnamaldehyde.
Rhizome is cytotoxic. Tuber is stimulant, expectorant, diuretic and carminative .
TULSI
Tulasi is perhaps the most sacred plant of India nest to kamala – the lotus. Particularly, the
leaves of Tulasi are used to worship Visnu. It is interesting to note that the plants sacred to
Sankara, Ganesha and Visnu are bilva, durva and tulasi, which alleviate vata, pitta and kapha
doshas, respectively. There are innumerable references of Tulasi in Indian mythology. The
ancient Ayurvedic scriptures have mentioned the plant in the management of several
diseases. The plant is supposed to have a purifying influence by liberating ozone and also is
said to repel the mosquitoes. Thus, in many parts of India, the plant is grown in the courtyard,
traditionally and worshipped daily as a necessary ritual for family will-being. Ayurvedic texts
categorise it as kasaghna – alleviates cough, svedala – induces sweating, ajirna nasaka –
mitigates indigestion and agnimandya nasaka – alleviates anorexia (Bhavaprakas)
The plant grows all over India up to 2000 meters height. It is grown in houses, temples and
gardens. An erectannual grows 0.5-1.5 meters in height and has red or purple quadrangular
branches. The leaves are opposite, about 2-4 cm long, margins entire or toothed, hairy on
both the surfaces, dotted with minute glands and are aromatic. The flowers tiny, purple and
inflorescence is a long spike or 12-14 cm in length. The fruits are small, smooth nut lets,
reddish grey in color.
Botanically, Tulasi is known as Ocimum sanctum and it belongs to family Lamiaceae. The
leaves contain an essential oil, which contains eugenol, eugenal, carvacrol, methylchavicol,
limatrol and caryophylline. The seeds contain oil composed of fatty acids and sitosterol. The
roots contain sitosterol and three triterpenes A, B, and C. The leaves also contain a steroid
ursolic acid and n-triacontanol. Eugenol (70.5), its methyl ether (4.8), nerol (6.4),
caryophyllene (7.5), terpinen –4-(0.4), decylaldehyde (0.2), selinene (0.4), pinene (0.4),
camphene (2.0) and a-pinene (3.5%) identified in essential oil by GC.
Tulasi is pungent and bitter in taste, pungent in the post digestive effect and has hot potency.
It alleviates kapha and vata doshas, but slightly aggravates the pitta dosha. It possesses light
and dry attributes. On the contrary the seeds are oily and slimy in attributes and have a cold
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potency. Tulasi is a stimulant, aromatic herb and effectively reduces the fever.
(Bhavaprakash)
The seeds, leaves and the roots of Tulasi have great medicinal value. It is used both,
internally as well as externally. Tulasi has mild antiseptic, analgesic properties and it relieves
the swellings also. Hence, it beneficial, externally, in various skin diseases. The paste of
leaves works well, with marica powder, when applied topically in ringworm infestations. The
dressing with the pulp of its leaves effectively controls the infections and hastens the healing
of chronic infected wounds. The leaves when chewed mitigate the infections of the gums.
Instillation of fresh juice of the leaves into ears is an effective domestic medicament for ear
aches. The massage with the leaves juice improves the circulation beneath the skin and
augments the sensation in the skin. In the headache due to sinusitis, the instillation of juice in
the nose facilitates the secretions of kapha and relieves the headache. The dried powder of the
leaves can be inhaled, like a snuff, for the same purpose.
Internally, Tulasi is used on many occasions, as a vehicle or an adjuvant. It enhances the
bioavailability of the medicine or acts synergistically, respectively. It is also used in number
of Ayurvedic proparations for bhavana, to potentate their effects, namely in Tribhuvana kurti,
Caturbhuja rasa etc. Tulsi is salutary to increase the appetite and improve digestion. It has a
mild laxative as well as vermicidal action, hence is rewarding in worm infestations. It is a
good blood purifier and is helpful in the diseases of the blood and heart. A tea prepared with
the leaves of Tulasi is a common domestic remedy for cold, cough, milk indigestion,
diminished appetite and malaise.
Tulasi has specified actions on the respiratory system-pranavaha srotasa. It effectively
liquefies the phlegm due to its hot and sharp attributes. It gives excellent results in cough due
to kapha, allergic bronchitis, asthma and eosinophilia. Combined with honey, the juice works
well to control the hiccup. In tubercular cough, Tulasi is also beneficial. It is an effective
panacea for fever, especially of kapha type, while given with honey and marica fruit powder.
In such conditions it effectively controls colds and reduces pain. Tulasi juice works as
amapacana, meaning it digests and destroys ama – the toxins.
The seeds of tulasi being cool and sweet along with its slimy-(picchila) attribite, are
beneficial in dysuria. The khira or pudding, prepared from the seeds, is useful to boost the
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energy in debility, especially due to pitta dosha. In diarrhea associated with bleeding, the
seeds soaked overnight in water to which a teaspoon of sugar is added, and taken in the
morning.
ANTIMICROBIAL ACTIVITY
There is a continuous and urgent need to discover new antimicrobial compounds with diverse
chemical structures and novel mechanisms of action because there has been an alarming
increase in the incidence of new and re-emerging infectious diseases. Another big concern is
the development of resistance to the antibiotics in current clinical use. In recent years, drug
resistance to human pathogenic bacteria has been commonly reported from all over the world.
In the present scenario of emergence of multiple drug resistance to human pathogenic
organisms, this has necessitated a search for new antimicrobial substances from other sources
including plants. Higher plants produce hundreds to thousands of diverse chemical
compounds with different biological activities. The antimicrobial compounds produced by
plants are active against plant and human pathogenic microorganisms. It is expected that
plant extracts showing target sites other than those used by antibiotics will be active against
drug-resistant microbial pathogens.
Antibiotics provide the main basis for the therapy of microbial (bacterial and fungal)
infections. Since the discovery of these antibiotics and their uses as chemotherapeutic agents
there was a belief in the medical fraternity that this would lead to the eventual eradication of
infectious diseases. However, over use of antibiotics has become the major factor for the
emergence and dissemination of multidrug resistant strains of several groups of
microorganisms. The worldwide emergence of multidrug resistant Escherichia coli and many
other ß-lactamase producers has become a major therapeutic problem Thus, in light of the
evidence of rapid global spread of resistant clinical isolates, the need to find new
antimicrobial agents is of paramount importance. However, the past record of rapid,
widespread emergence of resistance to newly introduced antimicrobial agents indicates that
even new families of antimicrobial agents will have a short life expectancy .For this reason,
researchers are increasingly turning their attention to herbal products, looking for new leads
to develop better drugs against MDR microbe strains The antimicrobial efficacy attributed to
some plants in treating diseases has been beyond belief. It is estimated that local communities
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have used about 10% of all flowering plants on Earth to treat various infections, although
only1% have gained recognition by modern scientists. During the last ten years the pace of
development of new antimicrobial drugs has slowed down while the prevalence of
resistance(especially multiple) has increased astronomically .The increase in number of
antibiotic resistant bacteria is no longer matched by expansion in the arsenal of agents
available to treat infections. Literature reports and ethnobotanical records suggest that plants
are the sleeping giants of pharmaceutical industry. They mayprovide natural source of
antimicrobial drugs that will/or provide novel or lead compounds that may be employedin
controlling some infections globally.
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Review OF
LITERATURE
Ethanol extracts of 19 Malaysian traditional vegetables (locally known as ‘ulam’)
belonging to 15 families were screened for antimicrobial and cytotoxic activities. Seven
extracts, i.e. Anacardium occidentale, Garcinia atroviridis, Sesbania grandiflora, Barringtonia
racemosa, Polygonum minus, Kaempferia galanga and Etlingera elatior displayed cytotoxic
activity against the HeLa (human cervical carcinoma) cell line with CD 50 values in the
range of 10–30 µg/ml.( M.M. Mackeen et al ., 1997). Ocimum sanctum the sacred 'Tulsi'
finds diverse uses in the indigenous system of Medicine. The leaves of the plant have been
used as an expectorant, diaphoretic, anticancer, antihelminthic, antiseptic, analgesic and tonic
rejuvinator. Dry leaves are used in fungal infections, the fresh juice of the leaves are used in
the treatment of bronchitis, otitis media, and skin diseases. Antibacterial activity of ether
extract of the leaves against Escherichia coli, Staphylococcus aureus, and Mycobacterium
tuberculosis have been reported.( Geeta et al .,2001). The antimicrobial effects of extracts
of neem seed (Azadirachta indica A. Juss.) were investigated using microbial growth
inhibition assays.( E. Coventry et Al.,2001). The antibacterial activity of Karanj (Pongamia
pinnata) and Neem (Azadirachta indica) seed oil in vitro against fourteen strains of
pathogenic bacteria was assessed (BASWA Manjula et al.,2001 ). Fifty four plant extracts
(methanol and aqueous) were assayed for their activity against multi-drug resistant
Salmonella typhi. Strong antibacterialactivity was shown by the methanol extracts of Aegle
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marmelos, Salmalia malabarica, Punica granatum, Myristica fragrans, Holarrhena
antidysenterica, Terminalia arjuna and Triphal (mixture of Emblica of fi cinalis, Terminalia
chebula and Terminalia belerica) ( Rani P et al .,2004 )
Ocimum sanctum fixed oil showed good antibacterial activity against Staphylococcus
aureus, Bacillus pumilus and Pseudomonas aeruginosa, where S. aureus was the most
sensitive organism.(Singh PP et al ., 2005). Screening was done of some plants of importance
in the Ayurvedic system of traditional medicine used in India to treat enteric diseases.
Volatile oil of dried rhizome of Kaempferia galanga obtained by water distillation was
determined for its chemical components using gas . Antimicrobial activity of the volatile oil
was tested against various microbes using agar disc diffusion method (S Tewtrakul et
al.,2005). The immunomodulatory activity of the neem-bark extract has been reported. The
medicinal and industrial uses of various parts of neem tree and the compounds isolated have
been reviewed. (Wafaa A. Helmy et al ., 2007 ). .. The antibacterial activity of guava
(Psidium guajava) and neem (Azadirachta indica) extracts against 21 strains of foodborne
pathogens were determined. Guava and neem extracts showed higher antimicrobial activity
against Gram-positive bacteria compared to Gram-negative bacteria except for V.
parahaemolyticus, P. aeroginosa, and A. hydrophila.( M.D. Mahfuzul Hoque et al ., 2007).
Basil or tulasi (Ocimum sanctum L.) is considered as a sacred plant and its various medicinal
properties have been mentioned in ancient medicinal text, Ayurveda. Different parts of this
plant are used for treatment of various ailments. The activity of essential oils obtained from
fresh and dried leaves of O. sanctum against clinical isolates of enteric bacteria
i.e., Escherichia coli, Shigella sp. and Salmonella typhi and yeast (Candida albicans) was
compared.( S. Mondal et al .,2007)
For evaluating antibacterial activity, both aqueous and organic solvent methanol was used.
The plants screened were Ocimum sanctum, Jatropha gossypifolia, Boerhavia diffusa,
Azadirachta indica, Solidago virgaurea, and Commelina benghalensis. The antibacterial
activity was assessed against six bacterial strains--Pseudomonas testosteroni, Staphylococcus
epidermidis, Klebsiella pneumoniae, Bacillus subtilis, Proteus morganii, Micrococcus flavus.
( Nair R et al .,2007). Essential oil obtained by hydrodistillation and two different solvent
21
extractions(petroleum ether and ethanol) from five Zingiberaceae species: ginger (Zingiber
officinaleRoscoe.), galanga (Alpinia galanga Sw.), turmeric (Curcuma longa L.), kaempferia
(Boesenbergia pandurata Holtt.) and bastard cardamom (Amomum xanthioides Wall.) was
characterized,(Krittika Norajit et al.,2007). Based on local use of common diseases and
Ethnobotanical knowledge, an attempt has been made to assess the antibacterial properties of
selected medicinal plants.VIZ. Ocimum sanctum (Tulsi), Origanum majorana (Ram Tulsi),
Cinnamomum zeylanicum (Dalchini), and Xanthoxylum armatum (Timur), for potential
antibacterial activity against 10 medically important bacterial strains, namely Bacillus
subtilis, Bacillus cereus, Bacillus thuringiensis, Staphylococcus aureus, Pseudomonas spp,
Proteus spp, Salmonella Typhi, Escherichia coli, Shigella dysentriae, Klebsiella
pneumoniae. The antibacterial activity of ethanol extracts was determined by agar well
diffusion method.( Bishnu joshi et al ., 2008). The enhancement of
hydrophilicity of DC air and oxygen plasma treated cotton fabric and its
effect on the antimicrobial efficacy when treated with neem leaf extract (K
Vaideki et al .,2009).
Silver nanoparticles are known to have bactericidal effects. A new generation of dressings
incorporating antimicrobial agents like silver nanoparticles is being formulated to reduce or
prevent infections. Apart from being environmentally friendly process, use of Neem leaves
extract might add synergistic antibacterial effect of Neem leaves to the biosynthesized
nanoparticles. With this hypothesis the biosynthetic production of silver nanoparticles by
aqueous extract of Neem leaves and its bactericidal effect in cotton cloth against E. Coli were
studied (Tripathi et al.,2009).Neem (Azadirachta indica) is an Indian tree well known for its
several pharmacological activities, including antimicrobial activity. More than 300
composites have already been isolated and azadirachtin (AZA) is its main active component.
Neem leaves hydroalcoholic extracts were prepared by percolation in 96% ethanol different
concentrations (50%, 60%, 70%, 80% and 90% (v/v)).( Priscila D. Alves et al .,2009).
In recent years this interest to evaluate plants possessing antibacterial activity for various
diseases is growing . Based on local use of common diseases and Ethnobotanical knowledge,
22
an attempt has been made to assess the antibacterial properties of selected medicinal
plants.VIZ. Ocimum sanctum (Tulsi), Origanum majorana (Ram Tulsi),Cinnamomum
zeylanicum (Dalchini), and Xanthoxylum armatum (Timur), for potential antibacterial activity
against 10 medically important bacterial strains, namely Bacillus subtilis, Bacillus cereus,
Bacillus thuringiensis, Staphylococcus aureus,
Pseudomonas spp, Proteus spp, Salmonella Typhi, Escherichia coli, Shigella dysentriae,
Klebsiella pneumonia (Bishnu Josh et al.,2009). The ethnobotanical efficacy of Indian
medicinal plants; Achyranthes aspera, Artemisia parviflora, Azadirachta indica, Calotropis
gigantean, Lawsonia inermis, Mimosa pudica, Ixora coccinea, Parthenium hysterophorus
and Chromolaena odorata were examined using agar disc diffusion method against clinical
bacteria (Escherichia coli and Staphylococcus aureus) and phytopathogenic bacteria
(Xanthomonas vesicatoria and Ralstonia solanacearum). Leaves were extracted using
different solvents such as methanol, ethanol, ethyl acetate and chloroform.( S. L. Sukanya et
al.,2009). Tulsi (Ocimum sanctum) extract has an antimicrobial activity against
Streptococcus mutans and the concentration of Tulsi (Ocimum sanctum) extract among the
15 concentrations investigated has the maximum antimicrobial activity was determined .
(Agarwal P et al.,2010).
. Tulsi has been tested against a variety of microorganisms like Candida albicans,
Staphyloccus aureus, enteric pathogens, Klebisella, Escherichia coli and Proteus. It has also
demonstrated antigonorrheal efficacy against multiresistant strains of Neisseria gonorrhea
and clinical isolates of beta lactamase-producing methicillin-resistant Staphylococcus aureus.
( Pooja Agarwal et al .,2010). Shoots and roots were induced from axillary buds of
Kaempferia galanga when cultured on Murashige and Skoog (MS) medium supplemented
with NAA + BAP (0.1mg + 1.0 mg/l). Liquid detergent (2%) + Alcohol (70%) + Mercuric
chloride (0.1%) used for surface sterilization of explants.Rhizomes were developed after four
months of transferring in to earthen pots. The methanolic extracts of both micropropagated
plant rhizome and natural plant rhizome were screened for antimicrobial activities against
two gram ‐ve and two gram +ve pathogenic bacteria i.e. Escherichia coli,Salmonella typhi,
23
Bacillus subtilis and Staphylococcus aureus.( HANUMANTHARAJU N et al.,2010). Four
medicinal plants (Quercus infectoria, Kaempferia galanga, Coptis chinensis and Glycyrrhiza
uralensis) as well as one traditional Thai treatment for aphthous ulcers based on these four
plants were tested for antimicrobial activity.( Mekseepralard C et al.,2010). Four medicinal
plants (Quercus infectoria, Kaempferia galanga, Coptis chinensis and Glycyrrhiza glabra) as
well as one traditional Thai treatment for aphthous ulcers based on these four plants were
tested for antimicrobial activity. MIC values for a range of bacteria and Candida albicans
were determined, with both type strains and clinical isolates being used. Antioxidant activity
was determined using the ABTS radical scavenging assay (Kamkaen et al.,2010).
Neem has been extensively used in Ayurveda, Unani and homoeopathic medicine and has
become a cynosure of modern medicine. The antibacterial activity of neem seed oil was
assessed in vitro against 14 strains of pathogenic bacteria. The present study was to find out
the antibacterial activity of neem leaves against human pathogenic bacteria, including
Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Micrococcus luteus, Bacillus
subtilis, Enterococcus faecalis and Streptococcus faecalis.( Saradhajyothi KOONA et
al.,2011). The antibacterial activity of aqueous extracts , chloroform extract ,alcohol extract
and oil obtained from leaves of Ocimum sanctum against the selected bacteria was observed (
Poonam Mishra et al .,2011). Antimicrobial activity in leaf extract of neem (Azadirachta
indica) against human pathogenic bacteria. E.coli, Staphylococcus aureus, Pseudomonas
aeruginosa , Salmonella typhimurium, Bacillus pumilus. Antimicrobial activities of alcoholic
extracts of neem leaves were used.( Maragathavalli, S et al.,2012). Kaempferia galanga L.
(KG), commonly known as cekor is one of those precious medicinal herbs of Zingiberaceae
that are still included in un-utilized herbs inspite of the variety of useful pharmacological
properties it possesses. Extracts of KG have anti-inflammatory, analgesic, nematicidal,
mosquito repellent, larvicidal, vasorelaxant, sedative, antineoplastic, antimicrobial, anti-
oxidant, antiallergic and wound healing properties.( Muhammad Ihtisham Umar et al.,2011).
Volatile oil from the rhizomes of Kaempferia galanga was isolated and characterized by Gas
Chromatography–Mass Spectroscopy. The antimicrobial activity of oil was tested against
human and plant pathogenic bacteria and fungi. (P.A. Mary Helen et al .,2011).
24
25
MATERIALS AND
METHODS
MATERIALS
COLLECTION OF PLANT MATERIAL
Fresh leaves of three different plants Neem ( Azadirachta indica A. Juss ) , Kacholam (
Kaempferia galanga L ), Tulsi ( Ocimum sanctum ) free from disease were collected from
different areas. The leaves were washed thoroughly 2-3 times with running water and once
26
with sterile distilled water. The material was chopped into small pieces and then air dried on a
sterile blotter under shade for 20-30 days.
PREPARATION OF PLANT EXTRACTS
10 g of each powdered plant material of Neem , Kacholam and Tulsi were defatted with
petroleum ether and extracted in a soxhlet extraction apparatus with 100% ethanol. The
solvent extracts were concentrated under reduced pressure and preserved at 5°C in airtight
bottle until further use.
TEST MICROORGANISMS USED FOR ANTIMICROBIAL STUDIES
Bacterial cultures of of both gram negative and gram positive - were obtained from the
culture collection centre. Different bacterial strains used for the antimicrobial studies were ,
Staphylococcus aureus (S.aureus) - It is a facultative anaerobic Gram -
positive coccal bacterium. It is frequently found as part of the normal skin flora on the skin
and nasal passages. S. aureus can cause a range of illnesses, from minor skin infections, such
as pimples, impetigo, boils(furuncles), cellulitis folliculitis, carbuncles, scalded skin
syndrome, and abscesses, to life-threatening diseases such
as pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock
syndrome (TSS), bacteremia, andsepsis. Its incidence ranges from skin, soft tissue,
respiratory, bone, joint, endovascular to wound infections. It is still one of the five most
common causes of nosocomial infections and is often the cause of postsurgical wound
infections.
Escherichia coli (E.coli) – it is a Gram-negative, rod-shaped bacteriumthat is commonly found
in the lower intestine of warm-blooded organisms (endotherms). Most E. coli strains are
harmless, but some serotypes can cause serious food poisoning in humans, and are
occasionally responsible for people making product recalls. The harmless strains are part of
the normal flora of the gut, and can benefit their hosts by producing vitamin K2, and by
preventing the establishment of pathogenic bacteria within the intestine.
27
Klebsiella pneumoniae – is a Gram-negative, non-
motile, encapsulated, lactose fermenting, facultative anaerobic, rod shaped bacterium found
in the normal flora of the mouth, skin, and intestines. It is clinically the most important
member of the Klebsiella genus of Enterobacteriaceae. K. pneumoniae can cause the
disease Klebsiella pneumonia. They cause destructive changes to human
lungs inflammation and hemorrhage with cell death (necrosis) that sometimes produces a
thick, bloody, mucoid sputum (currant jelly sputum). Typically these bacteria gain access
after a person aspirates colonizing oropharyngeal microbes into the lower respiratory tract.
Pseudomonas fluorescens (P.fluorescens) - is a common Gram-negative, rod-
shaped bacterium. It belongs to the Pseudomonas genus. P. fluorescens is an unusual cause of
disease in humans, and usually affects patients with compromised immune systems (e.g.,
patients on cancer treatment).
. Muller – Hinton agar
Beef infusion - 300 ml
Caesin hydrolysate - 17.5 g
Starch - 1.5g
Agar - 25 g
Distilled water – 1000 ml
The above constituents werw added one by one and the final volume was made up to 1000 ml
with distilled water . Then the pH was adjusted to 7 at 25◦Ccusing 0.1 Naoh/HCL . It was
autoclaved and was poured into sterile petridishes and was allowed to solidify . It was stored
at 4◦C.
Nutrient Broth
Nutrient broth was used for the preparation of inoculum
Peptone – 5g
28
Beef extract – 3g
Nacl – 5g
The above constituents were added one by one and the final volume was made up to 1000 ml
with distilled water . Then the pH was adjusted to 7 at 25◦c using0.1 NaoH / HCL. The
bacteria were maintained on nutrient broth (NB) at 37°C.
Streptomycin was used as a reference . It was purchased and was dissolved in 2ml of
distilled water.
DETERMINATION OF ANTIMICROBIAL ACTIVITY WITH WELL DIFFUSION
METHOD
In order to determine the biological significance and ability of the plant part, the antibacterial
activity was determined by agar well diffusion method. Muller Hinton agar plates (MHA)
were prepared as follows :
Overnight grown different species of bacteria such as E-coli, Staphylococcus aureus,
Klebsiella pneumoniae and Pseudomonas fluorescens were swabbed. Wells of approximately
10mm was bored used a well cutter and di concentrations of plant extracts (30,60µl ) was
added, the zone of inhibition was measured after overnight incubation and compared with
that of standard antibiotics, DMSO(Dimethylsulfoxide) was used as negative control and
streptomycin was used as reference.
The antimicrobial activity was determined by measuring the zone of inhibition after overnight
incubation and compared with standards.
29
RESULTS AND
DISCUSSION
30
RESULTS
The antimicrobial activity of the extracts and their potency was quantitatively assessed by the
presence or absence of inhibition zone and zone diameter,
respectively as given in Table 1.
Organism Sample Measurement of zone of
Inhibition(cm)
E Coli
Streptomycin
DMSO
2.1
1.1
31
Kacholam
Tulsi
Neem
1.3
1.4
1.2
Streptococcus
Streptomycin
DMSO
Kacholam
Tulsi
Neem
5.5
1.5
1.7
1.5
2.0
Staphylococcus
Streptomycin
DMSO
Kacholam
2.7
1.2
1.3
32
Tulsi
Neem
1.2
1.3
Klebsiella
Streptomycin
DMSO
Kacholam
Tulsi
Neem
5.5
1.5
1.7
1.5
2.0
33
DISCUSSION
34
Plants are important source of potentially useful structures for the development of new
chemotherapeutic agents. Many reports are available on the antiviral, antibacterial, antifungal,
anthelmintic, antimolluscal and anti-inflammatory properties of plants. Some of these
observations have helped in identifying the active principle responsible for such activities and
in the developing drugs for the therapeutic use in human beings. Emergence of multi-drug
resistance in human and animal pathogenic bacteria as well as undesirable side effects of
certain antibiotics has triggered immense interest in the search for new
antimicrobial drugs of plant origin..
In the present study , antimicrobial activity of plant extracts was determined
by agar well diffusion assay using Streptomycin as a positive control. The antimicrobial
spectrum was evaluated against E. coli, Pseudomonas, Staphylococcus, Klebsiella. The zone
of inhibition was more in wells corresponding to extracts of Neem (Azadirachta indica )
when compared with other two extracts (Kaempferia galanga and Ocimum sanctum) . Study
suggests potent antibacterial activity of all three plant extract which can find its application in
therapeutic regimens, confirming the great potential of bioactive compounds and are useful for
rationalizing the use of this plant in primary health care. In vivo data may be helpful in determining
the real potential usefulness of this plant for the treatment of infectious diseases.
35
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